Cholesterols, triglycerides, and phospholipids which are major lipids in the living body are combined with apoproteins to form lipoproteins in blood. The lipoproteins are grouped into chylomicron, very low density lipoprotein (VLDL), low density lipoprotein (LDL), high density lipoprotein (HDL), and the like according to their physical properties. Among these lipoproteins, it is known that LDL is a causative factor for arteriosclerosis, whereas HDL exhibits an anti-arteriosclerosis effect.
Total cholesterols and total triglycerides in blood have been assayed for the purpose of preventing ischemic heart diseases or evaluating the effects of treatments for such diseases. Epidemiological studies have proved that the cholesterol level in LDL has a positive correlation with the frequency of arteriosclerosis occurrence, whereas the cholesterol level in HDL has a negative correlation with the frequency of arteriosclerosis occurrence. Importance of knowing fluctuations in each lipoprotein fraction such as LDL and HDL has been understood. Nowadays, apoprotein B and apoprotein A-I, which are protein components forming each lipoprotein, or cholesterol in each lipoprotein fraction is assayed.
As mentioned above, as the method of assaying lipids components in blood, a method of determining the total amount of specific lipid contained in all lipoproteins such as total cholesterol and total triglycerides and a method of determining cholesterols in specific lipoproteins such as LDL, HDL, and the like can be given.
Of these, the former method is based on the combination of an enzyme for the lipid component to be assayed and conditions enabling the reaction of the lipid component with the enzyme without regard to the type of lipoproteins (for example, a surfactant with low lipoprotein selectivity such as Triton X-100). The method enables assaying the lipid components to be assayed in all lipoproteins.
The latter method, on the other hand, is based on the combination of an enzyme for the lipid component to be assayed and specific conditions enabling the enzyme to react only with the lipid component to be assayed in specific lipoproteins. The method enables the assay of the lipid components to be assayed in the specific lipoprotein fractions.
These specific conditions have been conventionally established by a method of isolating the lipoproteins to be assayed from a sample by ultra centrifugation, electrophoresis, gel filtration, precipitation using a precipitation agent (precipitation method), or the like (the method is hereinafter referred to as “fractionation method”).
In recent years, a number of methods for establishing specific conditions without using the fractionation method (hereinafter referred to as “direct methods”) have been developed. Such methods include a method of utilizing the different reaction times of bile acid for each lipoprotein fraction in the determination of HDL cholesterols (Japanese Patent Publication No. 016720/1994), a method of reacting the lipoproteins to be assayed with an enzyme under the condition that the lipoproteins not to be assayed stay aggregated (Japanese Patent Application Laid-open Publication No. 242110/1994), a method of using a sugar compound (Japanese Patent No. 2653755), a method of using a modification enzyme (Japanese Patent No. 2600065), a method of using a surfactant which does not dissolve lipoproteins (Japanese Patent No. 2799835), a method of removing cholesterols in lipoproteins not to be assayed by a previous enzyme reaction (Japanese Patent Application Laid-open Publication No. 000299/1997), a method of using carrageenan (Japanese Patent Application Laid-open Publication No. 121895/1997), a method of using a surfactant having lipoprotein selectivity (Japanese Patent Application Laid-open Publication No. 056395/1999), a method of using Calixarene (WO 98/59068), and a method of using a phosphorus compound (Japanese Patent Application Laid-open Publication No. 116400/2000).
Some of these direct methods are used in practice in daily clinical tests due to their simple procedures as compared with the precipitation method involving complicated procedures. However, even the direct methods which have been put into practice have problems. For example, the method of adding a precipitation agent to the reagent has problems: interference of the aggregates on assay precision and a damage to measuring equipment such as clogged flow passages with the products produced from reaction of the precipitation agent with an alkaline detergent for washing measuring equipment. As for a method of using a modification enzyme, there are problems such as process control in enzyme modification procedures (quality control) and cost increase.
Accordingly, an object of the present invention is to provide a method of assaying lipid components in blood and the like using a substance capable of establishing the specific conditions under the direct method while neither affecting assay precision nor damaging assay instruments, and also satisfying other conditions such as easy availability.